The present invention relates to novel methods for the preparation of alkylphosphonate nucleoside dimers, oligomeric compounds and methods of using such oligomeric compounds. In accordance with preferred embodiments of the invention, the present methods provide for the preparation of substantially diastereomerically pure (or enriched) alkylphosphonate compounds from substantially diastereomerically pure (or enriched) starting materials.
Modified oligonucleotides are of great value in molecular biological research and in applications such as anti-viral therapy. Modified oligonucleotides which can block RNA translation, and are nuclease resistant, are useful as antisense reagents. One such modification is the alkylphosphonate internucleotide linkage.
There are a number of properties associated with oligonucleotides having alkylphosphonate internucleoside linkages that are superior to native phosphodiester internucleotide linkages. The majority of research in this area has been with methylphosphonates (Miller et al., Antisense Research and Applications, 1993, pp. 189-203, Crooke, S. T. and Lebleu, B. (eds) CRC Press, Boca Raton). Oligonucleotides having methylphosphonate internucleotide linkages are isosteric to natural phosphate groups, have non-ionic backbones, are resistant to nucleases, have weak interactions with plasma proteins and can enter cells by passive diffusion or through absorptive endocytosis. Oligonucleotides having methylphosphonate internucleotide linkages are not substrates for RNase H when duplexed with RNA. When a chimeric oligonucleotide is prepared having a 2xe2x80x2-deoxy region flanked on either side by methylphosphonate regions, the resulting oligonucleotide is able to withstand intracellular nuclease degradation until bound to a target RNA. Upon formation of an oligonucleotide/target RNA duplex the 2xe2x80x2-deoxy region becomes a substrate for RNase H and thus can give rise to a terminating event.
In addition to chemical modifications of the internucleoside linkage, the chirality of each phosphorus containing internucleoside linkage also effects the ability of a modified oligonucleotide to bind to RNA. The standard oxidation of a phosphite to a phosphate internucleotide linkages results in attack on either side of the phosphorous atom resulting in Rp and Sp diastereomers. Modified phosphorus oligonucleotides are racemic mixtures having 2n isomers, with n equal to the number of intersugar linkages in the oligonucleotide. Thus, a 15-mer modified oligonucleotide, containing 14 asymmetric centers has 214 or 16,384 diastereomers. In view of this, in a racemic mixture, only a small percentage of the oligonucleotides are likely to specifically hybridize to a target mRNA or DNA with optimal affinity.
Backbone chirality may also affect the susceptibility of an oligonucleotide-RNA heteroduplex to RNase H activity. The ability to serve as a template for RNase H has significant therapeutic implications since it has been suggested that RNase H causes cleavage of the RNA component in an RNA-DNA oligonucleotide heteroduplex. For a variety of catalytic reactions, hydrolysis of the phosphodiester backbone of nucleic acids proceeds by a stereospecific mechanism (an in-line mechanism) and inversion of configuration. Therefore, there may be only a small percentage of oligonucleotides in a racemic mixture that contain the correct chirality for maximum hybridization efficiency and termination of translation. Thus, increasing the percentage of internucleotide linkages that are diastereomerically enriched that can serve as substrates for RNase H in a heteroduplex will likely lead to a more efficacious compound for antisense and other oligonucleotide therapies.
Oligonucleotides having pure Rp or Sp methylphosphonate internucleotide linkages have been prepared by a number of routes. The separation of a mixture of H-phosphonate diastereomers into pure Rp and Sp stereoisomers followed by alkylation with retention of configuration has given the corresponding pure Sp or Rp methylphosphonate (Seela, F., Kretschmer, U., J. Org. Chem., 1991, 56, 3861-3869). Another method requires the separation of a mixture of Rp and Sp 5xe2x80x2-O-protected-2xe2x80x2-O-deoxynucleosyl-3xe2x80x2-O-methylphosphinate (International Patent Application PCT/US93/06251, entitled xe2x80x9cTrivalent Synthesis of Oligonucleotides Containing Stereospecific Alkylphosphonates and Arylphosphonatesxe2x80x9d, filed Jun. 30, 1993), 3xe2x80x2-O-methylphosphonate (Jaworskamaslanka et al., Antisense and Nucleic Acid Drug Development, 1997, 7, 23-30), or -3xe2x80x2-O-methylphosphonothioate (International Patent Application PCT/US93/06277, entitled xe2x80x9cPentavalent synthesis of Oligonucleotides Containing Stereospecific Alkylphosphonates and Arylphosphonatesxe2x80x9d, filed Jun. 30, 1993) before their stereoselective incorporation in the oligonucleotide.
The incorporation of alkylphosphonate internucleoside linkages into modified oligonucleotide has been limited to only a few groups. The primary group that has been used is the methylphosphonate group. The use of benzylphosphonate internucleotide linkages is another group that has been synthesized (Eisenhardt et al., Nucleosides and Nucleotides, 1997, 16, 1669-1672).
The synthesis of a T-T dimer having a phosphorothioate internucleoside linkage has been previously reported using chiral auxiliary indole-oxazaphosphorine with 3xe2x80x2-OTBDPS-thymidine in the presence of several equivalents of 1,8-diazabicyclo[5.40]undec-7-ene (DBU) (Wang, J. C., Just, G., Tetrahedron Lett., 1997, 38, 3797-3800). After removal of the chiral auxiliary and sulfurization, the T-T phosphorothioate dimer is isolated in very high diastereomeric excess.
There remains a need for improved methods for preparing alkylphosphonates. There is further need for methods for preparing alkylphosphonates that are substantially diastereomerically pure or enriched. The present invention is directed to these, as well as other, important ends.
The present invention provides processes for preparing alkylphosphonate compounds having the structure: 
wherein each M1 and M2 independently, are hydrogen or a hydroxyl protecting group or one of M1 and M2 is hydrogen or a hydroxyl protecting group and the other of M1 and M2 is a covalent attachment to a solid support. In the foregoing structure, each J is, independently, hydrogen, a 2xe2x80x2-substituent group, or a protected 2xe2x80x2-substituent group and R1 is an electron withdrawing group. In such compounds, X2 is hydrogen or xe2x80x94(CH2)mxe2x80x94E2; E2 is hydrogen, alkyl, aryl, aralkyl, substituted aryl, a heterocycle, mixed heterocycle or heteroaryl and m is 0, 1, 2 or 3. Each Bx is a heterocyclic base moiety, preferably of the type associated with nucleobases, such as the preferred purines and pyrimidines. The methods comprise the steps of selecting a dimeric trialkylphosphite compound of the structure: 
and treating said dimeric trialkylphosphite compound, in a solvent, with a base under conditions of time temperature and pressure to form said alkylphosphonate compound.
It is preferred that one of M1 and M2 be a hydroxyl protecting group and the other of M1 and M2 be a covalent attachment to a solid support. In other preferred embodiments, both of M1 and M2 are hydroxyl protecting groups. It is preferred for some embodiments, that the electron withdrawing group be CN, COOR5 wherein R5 is C1-C8 alkyl, NO2, CF3, aryl, halogen or substituted aryl wherein said substituents are halogen, cyano, C1-C6 alkyl, perhalo C1-C3, alkyl, alkoxy, nitro, nitroso or carboxylate, with CN or NO2 being more preferred. In some preferred embodiments, X2 is H and R1 is CN.
In the processes of the invention, the alkylphosphonate compound is prepared as substantially a single diastereomer. It is also preferred that the dimeric trialkylphosphite compound be substantially diastereomerically pure as well.
In accordance with some embodiments, it has been found useful for the base to be 1,8-diazabicyclo[5.40]undec-7-ene (DBU), diisopropylethylamine (Hunig""s base), a secondary amine, piperidine, morpholine, or piperazine. It is preferred that the solvent be DMF, DMSO, THF, THP or CH3CN.
In accordance with other embodiments, the processes of the invention further comprise treating the alkylphosphonate compound with a chemical compound under conditions of time, temperature and pressure effective to reduce or modify said electron withdrawing group and optionally treating said alkylphosphonate compound having said reduced or modified electron withdrawing group with a chemical functional group having a reactive moiety thereon, to form an alkylphosphonate compound of the structure: 
wherein each M1 and M2 independently, are hydrogen or a hydroxyl protecting group or one of M1 and M2 is hydrogen or a hydroxyl protecting group and the other of M1 and M2 is a covalent attachment to a solid support. J is, independently, hydrogen, a 2xe2x80x2-substituent group or a protected 2xe2x80x2-substituent group, while R2 is a group having the formula xe2x80x94(CH2)mN(R3)(R4), xe2x80x94N(R3)(R4), or xe2x80x94C(O)N(R3)(R4). Each R3 and R4 independently, are H, C1-C10 alkyl, C1-C10 alkylamino, C1-C10 alkyl substituted alkylamino or a nitrogen protecting group; each X2 is hydrogen or xe2x80x94(CH2)mxe2x80x94E2; and E2 is hydrogen, alkyl, aryl, aralkyl, substituted aryl, a heterocycle, mixed heterocycle or heteroaryl. It is preferred that each m be, independently, 0, 1, 2 or 3; and that Bx be a heterocyclic base moiety.
In the foregoing processes, the chemical compound is preferably a reducing agent, especially BH3.SMe2, NaBH4 and CoCl2.6H2O, or LiBH4/MeOH and diglyme. In other embodiments, X2 is H and R2 is xe2x80x94CH2N(H)(R3) where R3 is C1-C10 alkyl. The chemical compound is preferably ammonia, a primary amine or a secondary amine wherein the substituents are C1-C10 alkyl, such as methylamine, dimethylamine, or dimethylaminoethlyamine.
The invention also features processes for preparing an alkylphosphonate compound of the structure: 
wherein
each M1 and M2 independently, are hydrogen or a hydroxyl protecting group or one of M1 and M2 is hydrogen or a hydroxyl protecting group and the other of M1 and M2 is a covalent attachment to a solid support. Preferably, each J is, independently, hydrogen, a 2xe2x80x2-substituent group, or a protected 2xe2x80x2-substituent group; R1 is an electron withdrawing group;
X2 is hydrogen or xe2x80x94(CH2)mxe2x80x94E2; and E2 is hydrogen, alkyl, aryl, aralkyl, substituted aryl, a heterocycle, mixed heterocycle or heteroaryl. M is preferably 0, 1, 2 or 3; and Bx is a heterocyclic base moiety. The processes comprise the steps of: selecting an H-phosphonate compound of the structure: 
xe2x80x83and treating said H-phosphonate compound with an xcex1,xcex2-unsaturated compound of the structure: 
xe2x80x83in a solvent under conditions of time temperature and pressure to form said alkylphosphonate compound. In preferred embodiments, the H-phosphonate compound is treated with a silylating agent under conditions of time temperature and pressure to form a silyl intermediate prior to said treatment with said xcex1,xcex2-unsaturated compound. Preferably, the silylating agent is N,O-bis(trimethylsilyl)trifluoroacetamide, trimethylsilyl chloride in pyridine, 1-trimethylsilyl imidazole, 3-TMS-2-oxazolidinone, 1-TMS-1,2,4-triazole, or Bis-TMS urea.
It is preferred that one of M1 and M2 be a hydroxyl protecting group and the other of M1 and M2 is a covalent attachment to a solid support, although both of M1 and M2 can be hydroxyl protecting groups. It is preferred that the alkylphosphonate compound be prepared as substantially a single diastereomer and that the trialkylphosphite, itself, be substantially diastereomerically pure.
Alkylphosphonate compound can also be prepared in accordance with the invention by starting with a cyclic phosphoramidite compound of the structure: 
wherein said E1 is covalently attached to P by a Nxe2x80x94P bond of said nitrogen containing heterocycle, nitrogen containing mixed heterocycle or nitrogen containing heteroaryl moiety to P. The cyclic structure is then treated with a base under conditions of time temperature and pressure to form an intermediate compound. The intermediate compound is then reacted with a further compound of the structure: 
under conditions of time, temperature and pressure to form the alkylphosphonate compound.
The present invention also provides compounds of the structure: 
wherein
Each M3 and M4 independently, are OH, a protected hydroxyl, a linkage to a solid support, an activated phosphate group, an activated phosphite group, a reactive group for forming an internucleotide linkage, a nucleotide, a nucleoside, or an oligonucleotide. Each J is, independently, hydrogen or a 2xe2x80x2-substituent group; R6 is an electron withdrawing group or a group having the formula xe2x80x94(CH2)nN(R3)(R4); each R3 and R4 independently, are H, C1-C10 alkyl or a nitrogen protecting group; and each X2 is hydrogen or xe2x80x94(CH2)nxe2x80x94E2. E2 is hydrogen, alkyl, aryl, aralkyl, substituted aryl, a heterocycle, mixed heterocycle or heteroaryl; n is 1, 2, or 3; and Bx is a heterocyclic base moiety.